This invention relates to electrophoresis equipment and, more particularly, to a method of applying alternating high voltage fields across an electrophoresis medium.
Electrophoretic methods make use of the difference in mobility between charged particles, suspended in a supporting medium and subject to the influence of an electrical field, to separate such particles from one another. The mobility of a charged particle is principally influenced by its charge-to-mass ratio but may be affected by a number of other factors including, importantly, the interference between the migrating particles and the structure of the supporting medium. Particles of similar charge-to-mass ratio may therefore be separated by exploiting the difference in their physical interaction with the support medium, which in turn is chiefly the result of differences in the migrating particles' size and shape.
If the electrical field to which the migrating particles are subjected may be varied, particles with similar charge-to-mass ratio may be separated by yet another means which takes advantage of differences between the particles relative ability to reorient themselves under a fluctuating electrical field.
Carle, et al., International Patent No. PCT/US 86/02038, discusses the separation of DNA molecules with similar charge-to-mass ratio through the use of pulsed or reversing electrical fields along one axis of the supporting medium. Carle, et al., postulate that such molecules, under a uniform field, orient themselves with respect to their migration, so as to have approximately equal mobility despite differences in their length. In an alternating field, however, the longer molecules are unable to adjust their orientation to the changing field as rapidly as the shorter molecules and hence cannot maintain a high mobility orientation. Separation of such molecules is obtained by alternating the electrical field across the supporting medium at the appropriate frequency to accentuate the difference in mobility between the longer and shorter molecules. The time period of one of the two polarities of voltage is adjusted to be longer than that of the reversed polarity insuring a net migration of molecules in one direction. An analogous procedure makes use of switched fields of different voltage rather than different periods.
Carle, et al., also disclose a means of using a pulsed rather than polarity switched electric field to separate certain molecules. Under this approach, the molecular separation results from an intrinsic propensity of the molecule to "relax" into lower mobility configurations in the absence of an applied field. The ability to separate molecules under this approach results from differences in "relaxation" time and reorientation from "relaxation" time between such molecules.
A different technique using alternating electrical fields across a supporting medium is disclosed in Cantor & Schwartz, U.S. Pat. No. 4,473,452 which discusses the application of two transverse alternating electrical fields along a plane of supporting medium. Such transverse fields may be of different voltages and may be varied in their angle to each other and may be pulsed or reversed in polarity.
The practical implementation of all of the above-described modulated field techniques requires one or more power sources that may be switched on and off or reversed in polarity automatically to provide a periodic variation in field intensity across the supporting medium. At the present time, these techniques involve total field voltages of less than several hundred volts and such power sources are constructed of a combination of a DC power supply of correct voltage and current rating in combination with a mechanical or solid state relay of a type commercially available. The relay is then actuated by a lower voltage timing module.
As modulated field electrophoresis techniques are developed, it is believed that voltages of several thousand volts or more will be required in order to realize two benefits. The first benefit of using such high voltages in these techniques is that for a given field gradient (expressed in volts per length of support medium) a higher voltage allows larger separation area which in turn may result in greater separation distances between migrating particles and hence improved resolution. The second benefit of higher electric fields is that the speed of migration of charged particles is approximately proportional to the strength of the gradient and therefore at high voltages the separation process may be greatly accelerated.
Nevertheless, there are significant obstacles to the use of high voltages, the most significant being the difficulty of reliably switching high DC voltages over many cycles. The prior art has made use of mechanical relays and has suggested the use of so called "solid state" relays.
The use of mechanical relays is severely limited in higher voltage applications as a result of increased propensity of higher voltage to arc across relay contacts during each relay cycle. Over many repetitions this arcing pits the relay contacts ultimately causing their failure. The expected lifetime of a mechanical reed relay operated at high voltages and switched once every several seconds may be less than one month. Some field modulated electrophoretic techniques require switching times as fast as once every several milliseconds.
Solid state relays solve the contact wear problem but are generally available only for relatively low voltages. Such solid state relays are typically composed of triggering circuitry, possibly including an optical isolator to allow a "floating voltage" trigger signal, and a solid state switch element, frequently a MOSFET. It should be noted that the commonly available and somewhat higher voltage Triac or SCR based solid state relays are intended for switching alternating currents and require the switched voltage to drop to zero before they will reset. These switches cannot be used in a electrophoresis design where DC voltages must be switched.
Commercially available solid state relays generally cannot be combined or "stacked" to handle higher voltages because of limitations of the driving circuitry. More precisely, the triggering circuitry on most D.C. solid-state relays is voltage referenced to one side of the switch so that if the switches are placed in series, the relay triggering voltage can no longer be precisely determined. Further, because of the tendency of such devices to switch asynchronously, an individual device in a stacked configuration may be subjected to many times its maximum rated voltage.
Solid state relays with "isolated" trigger circuitry may overcome this first obstacle to stacking, that imposed by the referencing of the trigger circuitry to the switch itself, but generally suffer from high trigger circuit impedances. Optically coupled devices are limited by the relatively high impedances of optically sensitive circuitry. Circuits triggered by capacitively or inactively isolated D.C. pulses, where the pulses toggle the relay on and off, are also high impedance circuits as a necessary result of the low energy transferred by a single pulse at practical voltage levels. Further, these pulse triggered circuits are necessarily sensitive to pulse-like capacitively coupled noise. The switching of high voltage fields across a supporting medium, in high voltage modulated field electrophoresis, by definition, involves the rapid switching of high voltages. This switching produces capacitively coupled high amplitude voltage spikes which makes susceptibility to electrical noise particularly acute in these applications. The polarized nature of most solid state switching devices and the need for reversing the polarity of the applied field in modulated field electrophoresis techniques requires that a number of solid state switches be connected to each other. This in turn, increases the possibility that any voltage spikes developed by one switch will be capacitively coupled into the switching trigger circuitry of another switch.
Finally, conventional solid state relays are generally designed to provide the maximum attainable speed of switching transition between the "on" state and the "off" state. This is to reduce the power dissipated in the solid state switching element and thereby increase the average current that may be handled by the device. Unfortunately, such rapid switching speeds increase the amplitude of voltage spikes and thereby increase the chance of false triggering of other switches in an electrophoresis application, and the chance of interference with other sensitive laboratory equipment.